In recent years, the development of alternative energy to oil is a very important issue, because of environmental problems, such as global warming and aerial pollution, in addition to the concern related to transportation energy supply. Plant biomass is the most abundant renewable energy source on earth, which is expected to serve as an alternative source to petroleum. The main components of plant biomass (lignocellulose) are polysaccharides such as celluloses and hemicelluloses (including xylan, arabinan and mannan), lignin and other pectins. These polysaccharides are hydrolyzed into monosaccharides such as glucose and xylose by a variety of glycoside hydrolases, and are used as a biofuel or a raw material of chemical products.
Lignocellulose having a complex structure is persistent, and is difficult to degrade or hydrolyze with a single enzyme. For this reason, the hydrolysis of cellulose among the polysaccharides generally requires three types of enzymes: an endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21) that are glycoside hydrolases. On the other hand, hemicelluloses include xylan, arabinan, mannan and the like, and although the composition thereof depends on the type of the plants, for example, xylan is a major constituent in broad-leaved trees, herbaceous plants and the like. For the hydrolysis of xylan, it is thought that xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) are required.
In the conventional lignocellulose to ethanol conversion process, high-solid loading up to 30-60% in initial substrate concentration has been attempted for the purpose of higher energy efficiency and less water usage. The enzymatic hydrolysis of lignocellulose by such high-solid loading processes results in the high viscosity of the hydrolyzed biomass solution so that the hydrolysis of lignocellulose hardly proceeds. Therefore, for example, by carrying out the enzymatic hydrolysis process at a high temperature of 80° C. or higher using a thermostable enzyme, in addition to an increase in the hydrolysis reaction rate, since the viscosity of the hydrolyzed biomass solution also reduces, the shortening of the hydrolysis reaction time and the reduction of the amount of enzyme are expected to be achieved. For this reason, for various glycoside hydrolases, development of enzymes that are more excellent in terms of thermostability has been desired.
Many thermostable glycoside hydrolases have been obtained by isolating and identifying the thermophilic microorganisms that live in a high temperature environment, cloning the genes from these cultured and isolated microorganisms and determining the DNA sequence thereof, followed by the expression thereof using Escherichia coli, filamentous fungi and the like. Numerous endoglucanases that can be used for hydrolysis of lignocellulose have been isolated from fungi, bacteria, and the like, and some of them are commercially available as reagents (for example, see Patent Documents 1 to 4 and Non-Patent Documents 1 to 5). However, many of them are endoglucanases having optimum temperatures within middle to high temperature ranges from 40° C. to 80° C., and few enzymes are capable of maintaining activity for a prolonged period of time in an ultra-high temperature range of 80° C. or higher. As the hyperthermostable endoglucanases having optimum temperatures of 80° C. or higher, endoglucanases derived from archaea (Patent Document 5, Non-patent Document 6), endoglucanases derived from bacteria (Patent Document 6, Non-Patent Document 7) and the like have been reported to date. However, the degradation characteristics for various substrate differ, and there is also a report describing the changes in the optimum temperature in a substrate-dependent manner. In addition, the thermal stability (in terms of the half-life of the enzyme activity) within an ultra-high temperature range is generally about 2 to 3 hours, which is not so high.